Novel selection ligands

ABSTRACT

Podocalyxin like proteins (e.g. PCLP and PCLP-2) having selectin ligand activity are provided. Also provided are nucleic acid compositions encoding novel PCLP-2 proteins. The subject polypeptide and nucleic acid compositions find use in a variety of applications, including research, diagnostic, and therapeutic agent screening applications, as well as in treatment therapies for disease conditions associated with podocalyxin like protein activity. In particular, methods of treating diseases associated with podocalyxin like protein selectin binding activity and/or chemokine presenting activity are provided, where such diseases include inflammation and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. §119 (e), this application claims priorityto the filing date of the U.S. Provisional Patent Application Serial No.60/074,389 filed Feb. 11, 1998 and U.S. Provisional Patent ApplicationSerial No. 60/111,663 filed Dec. 10, 1998, the disclosures of which areherein incorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant No.GM23547 awarded by the National Institute of Health. The Government hascertain rights in this invention.

INTRODUCTION

[0003] 1. Field of the Invention

[0004] The field of the invention is cell adhesion, particularlyselectin mediated cell adhesion, as well as the treatment of diseaseconditions related thereto.

[0005] 2. Background of the Invention

[0006] Selectin mediated binding plays an important and prominent rolein a variety of biological processes. Selectins are lectin like celladhesion molecules that mediate leukocyte-endothelial,leukocyte-leukocyte, leukocyte-platelet, platelet-endothelial andplatelet-platelet interactions. One critical biological process in whichselectin mediated binding plays a role is the maintenance of immunesurveillance.

[0007] Maintenance of immune surveillance depends on the constantrecirculation of lymphocytes from the blood through he vascular wallinto the tissues and eventually back into the blood. Lymphocyterecruitment from the blood into all secondary lymphoid organs (exceptthe spleen) as well as into many sites of chronic inflammation ismediated by a specialized post-capillary venule called a highendothelial venule. These vessels are defined by the distinct, cuboidalmorphology of their endothelial cells and their luminal presentation ofligands for the leukocyte adhesion molecule, L-selectin. Thislectin-like adhesion molecule is expressed on all classes of leukocytesin the blood and is responsible for the initial tethering and rolling ofa leukocyte on the endothelium prior to subsequent integrin mediatedfirm arrest and transmigration.

[0008] Several selectin ligands have, to date, been identified. TheL-selectin endothelial ligands in mouse that have been identified are:CD34, GlyCAM-1, MAdCAM-1 and sgp200. In addition, PSGL-1 has beenidentified as a leukocyte ligand for P-, E-, and L-selectin. Endothelialligands for L-selectin in humans are still poorly defined. However,human CD34 has been shown through a variety of assays to have selectinligand activity.

[0009] Although selectin mediated binding events play a critical role innormal physiological processes, disease conditions do exist for which itis desired to regulate or modulate, e.g. limit or prevent, the amount ofselectin mediated binding that occurs. Such conditions include: acute orchronic inflammation; autoimmune and related disorders, tissue rejectionduring transplantation, atherosclerosis, restenosis followingangioplasty, damaging thrombotic events, and the like.

[0010] As the above conditions all result from selectin mediated bindingevents, there is great interest in the identification of selectinligands and the elucidation of the mechanisms underlying such bindingevents. There is also great interest in the identification of treatmentmethodologies for these and related disease conditions, as well theidentification of active agents for use therein.

[0011] As such, there is continued interest in the identification of newselectin ligands and the elucidation of their role(s) in selectinmediated binding events, as well as the development of therapies fordisease conditions arising from such binding events.

Relevant Literature

[0012] U.S. Pat. No. 5,705,632 describes a podocalyxin like proteinexpressed on glomular epithelial cells. Sassetti et al., J. Exp. Med.(Jun. 15, 1998) 187:1965-1975 discloses the role of human PCLP as aselectin ligand. Other references describing podocalyxin and homologuesthereof include: Kershaw et al., J. Biol. Chem. (Jun. 20, 1997) 272:15708-15714; Kershaw et al., J. Biol. Chem. (Dec. 8, 1995) 270:29439-29446; McNagny et al., J. Cell Biol. (Sep. 22, 1997) 138:1395-1407; and Keiaschki et al., J. Cell Biol. (1984) 98:1591-1596. Hub& Rot, Am. J. of Pathology (March 1998) 152:749-757 and Middlet et al.,Cell (Oct. 31, 1997) 91:385-395 provide discussions of the role ofchemokines in leukocyte trafficking.

[0013] References providing background information on L-selectin bindinginclude: Lasky et al., Cell (Jun. 12, 1992) 69:927-938; Baumhueter etal., Science (Oct. 15, 1993) 262: 436-438; Girard & Springer, ImmunologyToday (1995) 16: 449; Rosen & Bertozzi, Current Opinion in Cell Biology(1994) 6: 663-673; Celi et al., Seminars in Hematology (1997) 34:327-335; as well as U.S. Pat. No. 5,580,862.

SUMMARY OF THE INVENTION

[0014] Podocalyxin like proteins having selectin ligand activity (e.g.PCLP, PCLP-2), as well as methods for producing the same, are provided.Also provided are nucleic acid compositions encoding novel polypeptideproducts with selectin ligand and/or chemokine presenting activity. Thesubject polypeptide and nucleic acid compositions find use in a varietyof applications, including research applications (e.g. in assaysdesigned to elucidate the biological mechanisms of cell adhesion andtrafficking), diagnostic applications, and therapeutic agent screeningapplications, as well as in therapeutic applications. Also provided aremethods of treating disease conditions associated with selectin mediatedbinding events, such as acute and chronic inflammation, autoimmunediseases and related disorders, tissue rejection and the like.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 provides the amino acid sequence (SEQ ID NO:02) and thenucleic acid sequence (SEQ ID NO:01) of human PCLP-2.

[0016]FIG. 2 provides the alignment of the PCLP arid CD34 cytoplasmictails. Regions of greater than 50% homology are boxed. Potential proteinkinase (S/TX-R/K) and casein kinase II phosphorylation (S/T-X-X-D/E)sites are circled.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Novel polypeptides having selectin ligand and/or chemokinepresenting activity (e.g. PCLP, PCLP-2), as well as methods for theirpreparation, are provided. Also provided are nucleic acid compositionsencoding PCLP-2 polypeptides. The subject polypeptide and nucleic acidcompositions find use in a variety of applications, including research,diagnostic, and therapeutic agent screening applications, as well as intherapeutic applications. Also provided are methods of treating diseaseconditions associated with selectin mediated binding events, such asacute and chronic inflammation, autoimmune diseases and relateddisorders, tissue rejection and the like.

[0018] Before the subject invention is farther described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0019] In this specification and the appended claims, the singular forms“a,”, “an,” and “the” include plural reference unless the contextclearly dictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

POLYPEPTIDE COMPOSITIONS

[0020] Podocalyxin-like proteins having selectin binding activity and/orchemokine resenting activity are provided. The subject podocalyxin-likeproteins, e.g. PCLP, PCLP-2, etc., are a sialoproteins that have a mucindomain, i.e. they are sialomucins, and as such are highly glycosylatedglycoproteins that contain a region of predominantly O-linkedcarbohydrate chains that are linked to serine or threonine residues. Thesubject proteins are transmembrane proteins having the following fourdistinct regions: (1) a mucin domain; (2) a cysteine-rich domain; (3) atransmembrane domain; and (4) cytoplasmic domain. In addition, PCLP-2contains an amino terminal domain which differentiates it from PCLP.

PCLP

[0021] The mucin domain of PCLP is an approximately 290 residue longamino acid stretch present at the N-terminus of the protein and has ahigh content of serine, threonine and proline, by which is meant thatthe relative frequency of these specific amino acids exceeds thefrequency of the remaining naturally occurring amino acids by a factorof about 2 to 5. This domain contains no sequence repeats and has noobvious sequence homology to the mucin domain of the CD34. The mucindomain is further characterized by dense O-glycosylation. C-terminal tothe mucin domain is a cysteine rich region of about 120 amino acidresidues in length in which about 4 cysteine residues are present. Thisregion is believed to form a globular structure under physiologicalconditions. The cytoplasmic domain of the subject PCLP is approximately75 amino acid residues in length and has a 25% overall sequence identitywith the cytoplasmic region of CD34, and further comprises threestretches of over 50% sequence identity with the cytoplasmic region ofCD34. See FIG. 2. The subject PCLP can have the amino acid sequence ofrat podocalyxin (as described in Keijaschki et al., J. Cell Biol. (1984)98:1591-1596); chicken podocalyxin, i.e. thromomucin (see McMagny etal., J. Cell Biol. (1997) 138:1395-1407); rabbit and human podocalyxin,as described in U.S. Pat. No. 5,705,623, the disclosure of which isherein incorporated by reference, as well as of homologues thereof,where by homologue is meant a protein having at least about 40 %,usually at least about 50% and more usually at least about 60% aminoacid sequence identity to the human PCLP protein, excluding the mucindomain, as disclosed in U.S. Pat. No. 5,705,623 and Kershaw et al., J.Biol. Chem. (1997) 272: 15708-15714, as well as having a Genbankaccession no. of U9759.

PCLP-2

[0022] In PCLP-2, the mucin domain is an approximately 140 residue longamino acid stretch adjacent to the amino terminal domain (described ingreater detail infra) of the protein and has a high content of serine,threonine and proline, by which is meant that the relative frequency ofthese specific amino acids exceeds the frequency of the remainingnaturally occurring amino acids by a factor of about 2 to 5 (generallythese three residues make up from about 30 to 40 and usually about 35number % of residues in the mucin domain). The mucin domain is furthercharacterized by dense O-glycosylation. C-terminal to the mucin domainis a cysteine rich region of about 150 amino acid residues in length inwhich about 3 cysteine residues are present. This region is believed toform a globular structure under physiological conditions. Thecytoplasmic domain of the subject PCLP is approximately 80 amino acidresidues in length and has a 25% overall sequence identity with thecytoplasmic region of CD34, and further comprises three stretches ofover 50% sequence identity with the cytoplasmic region of CD34. Theamino terminal domain is approximately 170 amino acids in length and isfurther characterized by: (a) being rich in acidic residues, where thenumber % of acidic amino acid residues ranges from about 20 to 40%,usually from about 25 to 35% and in many embodiments is 30% or close to30%; (b) having two tyrosine residues which serve as potential tyrosinesulfation sites; and being modified by a glycosaminoglycan chain. Inaddition to the above features, the PCLP-2 protein has at least 5potential glycosaminoglycan (GAG) attachment sites (one of which is theabove mentioned site in the amino or N-terminal domain) and 3 potentialN-linked glycosylation sites. The protein generally has an N-terminalcleavable signal peptide of about 30 to 35 residues in length. As such,the expressed PCLP-2 is post-translationally processed to produce maturePCLP-2.

[0023] The PCLP-2 proteins range in length from about 500 to 800,usually from about 550 to 700 and more usually from about 550 to 650amino acid residues, and the projected molecular weight of the subjectproteins based solely on the number of amino acid residues in theprotein ranges from about 50 to 80, usually from about 55 to 75 and moreusually from about 60 to 65 kDa. As the subject PCLP-2 proteins areglycoproteins, the actual molecular weight of these proteins issubstantially higher than the above projected molecular weights,typically ranging from about 3 to 5 times higher than the projectedmolecular weight. As such, the actual molecular weight typically rangesfrom about 150 to 300 kDa, usually from about 150 to 250 kDa and moreusually from about 175 to 225 kDa, e.g. about 200 kDa.

[0024] Of particular interest in certain embodiments is the mouse PCLP-2protein, where the mouse PCLP-2 protein of the subject invention has anamino acid sequence encoded by the mouse PCLP-2 gene, described above.Of particular interest in other embodiments is the human PCLP-2 protein,where the human PCLP-2 protein of the subject invention has an aminoacid sequence that is substantially the same as, or identical to, thesequence appearing as SEQ ID NO:02, infra. By substantially the same asis meant a protein having a sequence that has at least about 80%,usually at least about 90% and more usually at least about 98% sequenceidentity with the sequence of SEQ ID NO:02, as measured by BLAST, supra.

[0025] In addition to the specific PCLP-2 proteins described above,homologs or proteins (or fragments thereof) from other species, i.e.other animal species, are also provided, where such homologs or proteinsmay be from a variety of different types of species, usually mammals,e.g. rodents, such as mice, rats; domestic animals, e.g. horse, cow,dog, cat; and humans. By homolog is meant a protein having at leastabout 35%, usually at least about 40% and more usually at least about60% amino acid sequence identity to the specific human PCLP-2 protein asidentified in SEQ ID NOS:02, where sequence identity is determined usingthe BLAST algorithm, supra.

Additional Features of the Subject Proteins

[0026] In addition to the above characteristics, the subject PCLP andPCLP-2 are further characterized by being capable of presenting denseclusters of sulfated and sialylated O-linked oligosaccharides toL-selectin. The subject proteins are capable of binding to E-, P-, orL-selectin, as well as recombinant L-selectin, as well as to the HEVspecific MECA-79 monoclonal antibody. As such, the subject proteins arepreviously unidentified members of the group of glycoproteinscollectively referred to by those of skill in the art as peripheral nodeaddressin (PNAd). The subject proteins are further characterized bybeing able to support tethering and rolling of lymphocytes underphysiological flow conditions in vitro. In addition to the abovefeatures, the N-terminal acidic domain of PCLP-2 is capable of bindingto certain chemokines or chemoattractant agents, where such agentsinclude: secondary lymphoid chemokine (SLC), MIP3α, and the like.

[0027] The subject podocalyxin like proteins of the subject invention(e.g. human PCLP, human PCLP-2, mouse PCLP-2 or homologs thereof) arepresent in a non-naturally occurring environment, e.g. are separatedfrom their naturally occurring environment. In certain embodiments, thesubject proteins ares present in a composition that is enriched for thesubject protein as compared to the subject protein in its naturallyoccurring environment. As such, purified PCLP and PCLP-2 are provided,where by purified is meant that the subject protein is present in acomposition that is substantially free of other, non-subject proteins,where by substantially free is meant that less than 90%, usually lessthan 60% and more usually less than 50% of the composition is made up ofother or non-subject (e.g. non-PCLP, non-PCLP-2) proteins.

[0028] In certain embodiments of interest, the subject proteins arepresent in a composition that is substantially free of the constituentsthat are present in its naturally occurring environment. For example, ahuman PCLP or PCLP-2 protein comprising composition according to thesubject invention in this embodiment will be substantially, if notcompletely, free of those other biological constituents, such asproteins, carbohydrates, lipids, etc., with which it is present in itsnatural environment. As such, protein compositions of these embodimentswill necessarily differ from those that are prepared by purifying theprotein from a naturally occurring source, where at least trace amountsof the protein's constituents will still be present in the compositionprepared from the naturally occurring source.

[0029] The subject proteins may also be present as an isolate, by whichis meant that the subject protein is substantially free of other,non-subject proteins and other naturally occurring biologic molecules,such as oligosaccharides, polynucleotides and fragments thereof, and thelike, where substantially free in this instance means that less than70%, usually less than 60% and more usually less than 50% of thecomposition containing the isolated protein is some other (e.g.non-PCLP, non-PCLP-2) naturally occurring biological molecule. Incertain embodiments, the subject protein is present in substantiallypure form, where by substantially pure form is meant at least 95%,usually at least 97% and more usually at least 99% pure.

[0030] In many preferred embodiments, the subject protein is present inits naturally glycosylated state, i.e. it will have the sameglycosylation pattern as that found in naturally occurring protein suchthat it is a glycoprotein. In other embodiments, the proteins arenon-naturally glycosylated. By non-naturally glycosylated is meant thatthe protein has a glycosylation pattern, if present, which is not thesame as the glycosylation pattern found in the corresponding naturallyoccurring protein. For example, human PCLP-2 of the subject inventionand of this particular embodiment is characterized by having aglycosylation pattern, if it is glycosylated at all, that differs fromthat of naturally occurring human PCLP-2. Thus, the non-naturallyglycosylated PCLP-2 proteins of this embodiment include non-glycosylatedPCLP-2 proteins, i.e. proteins having no covalently bound glycosylgroups.

[0031] In addition to the naturally occurring proteins, polypeptideswhich vary from the naturally occurring proteins are also provided, e.g.PCLP-2 polypeptides. By PCLP-2 polypeptides is meant proteins having anamino acid sequence encoded by an open reading frame (ORF) of a PCLP-2gene, described infra, including the full length PCLP-2 protein andfragments thereof, particularly biologically active fragments and/orfragments corresponding to functional domains, e.g. the N-terminaldomain, the mucin domain, etc.; and including fusions of the subjectpolypeptides to other proteins or parts thereof, e.g. immunoglobulindomains. Fragments of interest will typically be at least about 10 aa inlength, usually at least about 50 aa in length, and may be as long as300 aa in length or longer, but will usually not exceed about 1000 aa inlength, where the fragment will have a stretch of amino acids that isidentical to a PCLP-2 protein of SEQ ID NO:02, or a homolog thereof; ofat least about 10 aa, and usually at leas, about 15 aa, and in manyembodiments at least about 50 aa in length.

[0032] Also provided by the subject invention are novel ligands havingselectin binding activity. The term ligand, as used herein, refers toany compound capable of binding to a selectin receptor, particularly E-,P- and L-selectin, and as such includes proteins and peptides,oligosaccharides, and the like, as well as binding mimetics thereof,including small molecule binding mimetics thereof. The subject ligandsare capable of binding to selectin receptors in a manner analogous tothe binding activity of HEV derived PCLP or PCLP-2, and will generallycomprise the mucin like domain of HEV derived PCLP or PCLP-2, or thefunctional equivalent thereof. As such, the subject ligands in manyembodiments will typically at least comprise a region of dense O-linkedoligosaccharides covalently bonded to serine and threonine residues of apolypeptide having an amino acid sequence of at least about 30%, usuallyat least about 50% and more usually at least about 70% sequence identitywith the amino acid sequence of the mucin domain human PCLP or PCLP-2.

Preparation of the Subject Proteins

[0033] The subject proteins, where obtained from naturally occurringsources, are generally derived from selectin ligand presenting cells,including endothelial cells, leukocytes and platelets, where they arepreferably derived from endothelial cells of high endothelial venules(HEV) of mammalian secondary lymphoid organs, and more preferably fromhuman tonsilar HEV. The subject proteins may also be derived fromsynthetic means, e.g. by expressing a recombinant gene encoding theprotein, such as the polynucleotide compositions described infra, in asuitable host under conditions sufficient for post-translationalmodification to occur in a manner that provides the expressed proteinwith selectin binding activity. For expression, an expression cassettemay be employed. The expression vector will provide a transcriptionaland translational initiation region, which may be inducible orconstitutive, where the coding region is operably linked under thetranscriptional control of the transcriptional initiation region, and atranscriptional and translational termination region. These controlregions may be native to a the gene, or may be derived from exogenoussources.

[0034] Expression vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding heterologous proteins. A selectablemarker operative in the expression host may be present. Expressionvectors may be used for the production of fusion proteins, where theexogenous fusion peptide provides additional functionality, i.e.increased protein synthesis, stability, reactivity with definedantisera, an enzyme marker, e.g. β-galactosidase, etc.

[0035] Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

[0036] The subject proteins and polypeptides may be expressed inprokaryotes or eukaryotes in accordance with conventional ways,depending upon the purpose for expression. For large scale production ofthe protein, a unicellular organism, such as E. coli B. subrulis, S.cerevisiae, insect cells in combination with baculovirus vectors, orcells of a higher organism such as vertebrates, particularly mammals,e.g. COS 7 cells, may be used as the expression host cells. In somesituations, it is desirable to express the gene in eukaryotic cells,where the protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the complete proteinmay be used to identify and investigate parts of the protein importantfor function.

[0037] Once the source of the protein is identified and/or prepared,e.g. a transfected host expressing the protein is prepared, the proteinis then purified to produce the desired protein comprising composition.Any convenient protein purification procedures may be employed, wheresuitable protein purification methodologies are described in Guide toProtein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may be prepared from the original source, e.g.naturally occurring cells or tissues that express PCLP or PCLP-2 or theexpression host expressing PCLP-2 or PCLP, and purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,and the like.

Nucleic Acid Compositions

[0038] Also provided are nucleic acid compositions encoding PCLP-2proteins and polypeptides, as well as fragments thereof. By nucleic acidcomposition is meant a composition comprising a sequence of DNA havingan open reading frame that encodes a PCLP-2 polypeptide, i.e. a geneencoding a polypeptide having PCLP-2 activity, and is capable, underappropriate conditions, of being expressed as a PCLP-2 polypeptide. Alsoencompassed in this term are nucleic acids that are homologous,substantially similar or identical to the nucleic acids encoding PCLP-2polypeptides or proteins. Thus, the subject invention provides genesencoding mammalian PCLP-2, such as genes encoding human PCLP-2 andhomologs thereof and mouse PCLP-2 and homologs thereof.

[0039] The coding sequence of the human PCLP-2 gene, i.e. the human cDNAencoding the human PCLP-2 protein, has the nucleic acid sequenceidentified as SEQ ID NO:01, infra. The coding sequence of the mousePCLP-2 gene, i.e. the mouse cDNA encoding the mouse PCLP-2 protein, ischaracterized by having the following ESTs derived from it: AA049027,W13047, W36468, AA008836, W54261, AA208106, AA155174 (the ESTidentifiers are their Gene Bank accession numbers).

[0040] The source of homologous genes to those specifically listed abovemay be any mammalian species, e.g., primate species, particularly human;rodents, such as rats and mice, canines, felines, bovines, ovines,equines, etc; as well as non-mammalian species, e.g. yeast, nematodes,etc. Between mammalian species, e.g., human and mouse, homologs havesubstantial sequence similarity, e.g. at least 75% sequence identity,usually at least 90%, more usually at least 95% between nucleotidesequences. Sequence similarity is calculated based on a referencesequence, which may be a subset of a larger sequence, such as aconserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990), J. Mol. Biol.215:403-10 (using default settings), Unless indicated otherwise, thesequence similarity values reported herein were determined using theabove referenced BLAST program using default settings. The sequencesprovided herein are essential for recognizing PCLP-2 related andhomologous polynucleotides in database searches.

[0041] Nucleic acids encoding the PCLP-2 proteins and PCLP-2polypeptides of the subject invention may be eDNAs or genomic DNAs, aswell as fragments thereof. The term “PCLP-2-gene” shall be intended tomean the open reading frame encoding specific PCLP-2 proteins andpolypeptides, and PCLP-2 introns, as well as adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression, up to about 20 kb beyond the coding region, but possiblyfurther in either direction. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto a host genome.

[0042] The term “cDNA” as used herein is intended to include all nucleicacids that share the arrangement of sequence elements found in nativemature mRNA species, where sequence elements are exons and 3′ and 5′non-coding regions. Normally mRNA species have contiguous exons, withthe intervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding a PCLP-2protein.

[0043] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3′ and 5′untranslated regions found in the mature mRNA. It may further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

[0044] The nucleic acid compositions of the subject invention may encodeall or a part of the subject PCLP-2 proteins and polypeptides, describedin greater detail infra.

[0045] Double or single stranded fragments may be obtained from the DNAsequence by chemically synthesizing oligonucleotides in accordance withconventional methods, by restriction enzyme digestion, by PCRamplification, etc. For the most part, DNA fragments will be of at least15 nt, usually at least 18 nt or 25 nt, and may be at least about 50 nt.

[0046] The PCLP-2 genes of the subject invention are isolated andobtained in substantial purity, generally as other than an intactchromosome. Usually, the DNA will be obtained substantially free ofother nucleic acid sequences that do not include a PCLP-2 sequence orfragment thereof, generally being at least about 50%, usually at leastabout 90% pure and are typically “recombinant”, i.e. flanked by one ormore nucleotides with which it is not normally associated on a naturallyoccurring chromosome.

[0047] In addition to the plurality of uses described in greater detailin following sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the PCLP-2 polypeptides, asdescribed above.

Uses of the Subject Polypeptide and Nucleic Acid Compositions

[0048] The subject polypeptide and nucleic acid compositions find use ina variety of different applications, including research, diagnostic, andtherapeutic agent screening/discovery/preparation applications, as wellas the treatment of disease conditions associated with PCLP and/orPCLP-2 activity, e.g. selectin ligand activity and/or chemokinepresenting activity.

Research Applications

[0049] The subject nucleic acid compositions find use in a variety ofresearch applications. Research applications of interest include: (a)the identification of PCLP-2 homologs; (b) as a source of novel promoterelements; (c) the identification of PCLP-2 expression regulatoryfactors; (d) as probes and primers in hybridization applications, e.g.PCR; (e) the identification of expression patterns in biologicalspecimens; (f) the preparation of cell or animal models for PCLP-2function; (g) the preparation of in vitro models for PCLP-2 function;etc.

Identification of PCLP-2 Homologs

[0050] Homologs of PCLP-2 are identified by any of a number of methods.A fragment of the provided cDNA may be used as a hybridization probeagainst a cDNA library from the target organism of interest, where lowstringency conditions are used. The probe may be a large fragment, orone or more short degenerate primers. Nucleic acids having sequencesimilarity are detected by hybridization under low stringencyconditions, for example, at 50° C. and 6×SSC (0.9 M sodium chloride/0.09M sodium citrate) and remain bound when subjected to washing at 55° C.in 1×SSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequenceidentity may be determined by hybridization under stringent conditions,for example, at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5mM sodium citrate). Nucleic acids having a region of substantialidentity to the provided PCLP-2 sequences, e.g. allelic variants,genetically altered versions of the gene, etc., bind to the providedPCLP-2 sequences under stringent hybridization conditions. By usingprobes, particularly labeled probes of DNA sequences, one can isolatehomologous or related genes.

Identification of Novel Promoter Elements

[0051] The sequence of the 5′ flanking region may be utilized forpromoter elements, including enhancer binding sites, that provide forregulation in tissues where PCLP-2 is expressed. The tissue specificexpression is useful for determining the pattern of expression, and forproviding promoters that mimic the native pattern of expression.Naturally occurring polymorphisms in the promoter region are useful fordetermining natural variations in expression, particularly those thatmay be associated with disease.

Identification of PCLP-2 Expression Regulatory Factors

[0052] Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g. sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al. (1995), Mol. Med.1:194-205; Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin andRichard-Foy (1995), Eur. J. Biochem. 232:620-626.

[0053] The regulatory sequences may be used to identify cis actingsequences required for transcriptional or translational regulation ofPCLP-2 expression, especially in different tissues or stages ofdevelopment, and to identify cis acting sequences and trans-actingfactors that regulate or mediate PCLP-2 expression. Such transcriptionor translational control regions may be operably linked to a PCLP-2 genein order to promote expression of wild type or altered PCLP-2 or otherproteins of interest in cultured cells, or in embryonic, et al. or adulttissues, and for gene therapy.

Probes and Primers

[0054] Small DNA fragments are useful as primers for PCR, hybridizationscreening probes, etc. Larger DNA fragments, i. e. greater than 100 ntare useful for production of the encoded polypeptide, as described inthe previous section. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

Identification of Expression Patterns in Biological Specimens

[0055] The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of PCLP-2 gene expression in the sample.

The Preparation of PCLP-2 Mutants

[0056] The sequence of an PCLP-2 gene, including flanking promoterregions and coding regions, may be mutated in various ways known in theart to generate targeted changes in promoter strength, sequence of theencoded protein, etc. The DNA sequence or protein product of such amutation will usually be substantially similar to the sequences providedherein, i.e. will differ by at least one nucleotide or amino acid,respectively, and may differ by at least two but not more than about tennucleotides or amino acids. The sequence changes may be substitutions,insertions, deletions, or a combination thereof. Deletions may furtherinclude larger changes, such as deletions of a domain or exon. Othermodifications of interest include epitope tagging, e.g. with the FLACsystem, HA, etc. For studies of subcellular localization, fusionproteins with green fluorescent proteins (GFP) may be used.

[0057] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site specific mutagenesis may be found inGustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene37:111-23; Colicelli et al. (1985), Mol. Gen. Genet. 199:537-9; andPrentki et al. (1984), Gene 29:303-13. Methods for site specificmutagenesis can be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al.(1993), Gene 126:35-41; Sayers et al. (1992), Biotechniques 13:592-6;Jones and Winistorfer (1992), Biotechniques 12:528-30; Barton et al.(1990), Nucleic Acids Res 18:7349-55; Marotti and Tomich (1989), GeneAnal. Tech. 6:67-70; and Zhu (1989), Anal Biochem 177:120-4. Suchmutated genes may be used to study structure-function relationships ofPCLP-2, or to alter properties of the protein that affect its functionor regulation.

Production of In Vivo Models of PCLP-2 Function

[0058] The subject nucleic acids can be used to generate transgenic,non-human animals or site specific gene modifications in cell lines.Transgenic animals may be made through homologous recombination, wherethe normal PCLP-2 locus is altered. Alternatively, a nucleic acidconstruct is randomly integrated into the genome. Vectors for stableintegration include plasmids, retroviruses and other animal viruses,YACs, and the like.

[0059] The modified cells or animals are useful in the study of PCLP-2function and regulation. For example, a series of small deletions and/orsubstitutions may be made in the host's native PCLP-2 gene to determinethe role of different exons in cholesterol metabolism, e.g. cholesterolester synthesis, cholesterol absorption, etc. Specific constructs ofinterest include anti-sense PCLP-2, which will block PCLP-2 expression,expression of dominant negative PCLP-2 mutations, and over-expression ofPCLP-2 genes. Where a PCLP-2 sequence is introduced, the introducedsequence may be either a complete or partial sequence of a PCLP-2 genenative to the host, or may be a complete or partial PCLP-2 sequence thatis exogenous to the host animal, e.g., a human PCLP-2 sequence. Adetectable marker such as lac Z, may be introduced into the pclp-2locus, where upregulation of PCLP-2 expression will result in an easilydetected change in phenotype.

[0060] One may also provide for expression of the PCLP-2 gene orvariants thereof in cells or tissues where it is not normally expressed,at levels not normally present in such cells or tissues, or at abnormaltimes of development.

[0061] DNA constructs for homologous recombination will comprise atleast a portion of the PCLP-2 gene native to the species of the hostanimal, wherein the gene has the desired genetic modification(s), andincludes regions of homology to the target locus. DNA constructs forrandom integration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included, Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.(1990), Meth. Enzymol. 185:527-537.

[0062] For embryonic stem (ES) cells, an ES cell line may be employed,or embryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

[0063] The chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogeneic or congenic grafts or transplants, or in in vitro culture.The transgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on PCLP-2 activity.

Production of In Vitro Models of PCLP-2 Function

[0064] Also provided by the subject invention are in vitro models ofPCLP and PCLP-2 function, e.g. the role of PCLP or PCLP-2 as a selectinligand, the role of PCLP-2 as a chemokine presenter, etc. In in vitromodels of the selectin ligand function of PCLP or PCLP-2, binding eventsbetween the podocalyxin like protein and a selectin receptor aremodulated, e.g. inhibited. The selectin receptor will generally be areceptor which binds to PCLP-2 under physiological conditions and is amember of the selectin family of receptors that have an amino terminalC-type lectin domain followed by an EFG-like domain, a variable numberof short consensus repeats known as SCR, CRP or sushi domains, and sharegreater than 50% homology in their lectin and EFG domains. Of particularinterest is the modulation of PCLP-2 selectin binding events in whichthe selectin is L-, P-, or E-selectin. In in vitro methods of inhibitingselectin mediated binding events, such methods typically includecontacting a selectin receptor with HEV derived podocalyxin likeprotein, e.g. PCLP or PCLP-2, and a competitor or inhibitor underconditions sufficient for selectin podocalyxin like protein binding tooccur. The competitor may be any compound that is, or is suspected tobe, a compound capable of specifically binding to selectin, where ofparticular interest in many embodiments is the use of the subjectligands described above as competitors. Depending on the particularmethod, one or more of, usually one of, the specified components may belabeled, where by labeled is meant that the components comprise adetectable moiety, e.g. a fluorescent or radioactive tag, or a member ofa signal producing system, e.g. biotin for binding to anenzyme-5treptaviin conjugate n which the enzyme is capable of convertinga substrate to a chromogenic product.

[0065] The above in vitro methods may be designed a number of differentways, where a variety of assay configurations and protocols may beemployed, as are known in the art. For example, one of the componentsmay be bound to a solid support, and the remaining components contactedwith the support bound component. The above components of the method maybe combined at substantially the same time or at different times, e.gsoluble L-selectin and ligand may be combined first, and the resultantmixture subsequently combined with substrate bound HEV derived PCLP orPCLP-2. Following the contact step, the subject methods will generally,though not necessarily, further include a washing step to remove unboundcomponents, where such a washing step is generally employed whenrequired to remove label that would give rise to a background signalduring detection, such as radioactive or fluorescently labelednon-specifically bound components. Following the optional washing step,the presence of bound selectin-PCLP or selectin-PCL-P-2 complexes willthen be detected.

[0066] A variety of assay formats may also be employed for elucidatingthe role of PCLP-2 as a presenting molecule for chemokines. For example,an ELISA format can be employed in which the N-terminal domain of PCLP-2is stably associated with the surface of a solid support. The supportbound PCLP-2 is then contacted with tagged chemokine to determinewhether the chemokine binds to the N-terminal domain. Inclusion of asecond chemokine under a competitive format allows the testing of aplurality of different chemokines for their ability to bind to PCLP-2.From the resultant data, the chemokine presenting functionality ofPCLP-2 can readily be determined.

[0067] The above described in vitro methods find particular use inassays designed to obtain information regarding cell adhesion inmammals, particularly PCLP/selectin or PCLP-2/selectin mediated celladhesion, where such cell adhesion includes cell adhesion resultingfrom: leukocyte-endothelium interactions; leukocyte-leukocyteinteractions; platelet-leukocyte interactions; platelet-endothelialinteractions; and platelet-platelet interactions. The above describedassays also find use in obtaining information regarding cellularemigration, e.g. leukocyte emigration. The above described in vitromethods also find use in screening assays designed to identify compoundsthat inhibit the binding of selectins to PCLP or PCLP-2.

Diagnostic Applications

[0068] Also provided are methods of diagnosing disease states associatedwith PCLP-2 activity, e.g. based on observed levels of PCLP-2 or theexpression level of the PCLP-2 gene in a biological sample of interest.Samples, as used herein, include biological fluids such as semen, blood,cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like;organ or tissue culture derived fluids; and fluids extracted fromphysiological tissues. Also included in the term are derivatives andfractions of such fluids. The cells may be dissociated, in the case ofsolid tissues, or tissue sections may be analyzed. Alternatively alysate of the cells may be prepared.

[0069] A number of methods are available for determining the expressionlevel of a gene or protein in a particular sample. Diagnosis may beperformed by a number of methods to determine the absence or presence oraltered amounts of normal or abnormal PCLP-2 in a patient sample. Forexample, detection may utilize staining of cells or histologicalsections with labeled antibodies, performed in accordance withconventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies of interest are added to the cell sample, andincubated for a period of time sufficient to allow binding to theepitope, usually at least about 10 minutes. The antibody may be labeledwith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabels for direct detection. Alternatively, a second stage antibody orreagent is used to amplify the signal. Such reagents are well known inthe art. For example, the primary antibody may be conjugated to biotin,with horseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to aflourescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

[0070] Alternatively, one may focus on the expression of PCLP-2.Biochemical studies may be performed to determine whether a sequencepolymorphism in an PCLP-2 coding region or control regions is associatedwith disease. Disease associated polymorphisms may include deletion ortruncation of the gene, mutations that alter expression level, thataffect the activity of the protein, etc.

[0071] Changes in the promoter or enhancer sequence that may affectexpression levels of PCLP-2 can be compared to expression levels of thenormal allele by various methods known in the art. Methods fordetermining promoter or enhancer strength include quantitation of theexpressed natural protein; insertion of the variant control element intoa vector with a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

[0072] A number of methods are available for analyzing nucleic acids forthe presence of a specific sequence, e.g. a disease associatedpolymorphism. Where large amounts of DNA are available, genomic DNA isused directly. Alternatively, the region of interest is cloned into asuitable vector and grown in sufficient quantity for analysis. Cellsthat express PCLP-2 may be used as a source of mRNA, which may beassayed directly or reverse transcribed into cDNA for analysis. Thenucleic acid may be amplified by conventional techniques, such as thepolymerase chain reaction (PCR), to provide sufficient amounts foranalysis. The use of the polymerase chain reaction is described inSaiki, et al. (1985), Science 239:487, and a review of techniques may befound in Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSHPress 1989, pp.14.2-14.33. Alternatively, various methods are known inthe art that utilize oligonucleotide ligation as a means of detectingpolymorphisms, for examples see Riley et al. (1990), Nucl. Acids Res.18:2887-2890; and Delahunty et al. (1996), Am. J. Hum. Genet.58:1239-1246.

[0073] A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ¹²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated Lo biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

[0074] The sample nucleic acid, e.g. amplified or cloned fragment, isanalyzed by one of a number of methods known in the art. The nucleicacid may be sequenced by dideoxy or other methods, and the sequence ofbases compared to a wild-type PCLP-2 sequence. Hybridization with thevariant sequence may also be used to determine its presence, by Southernblots, dot blots, etc. The hybridization pattern of a control andvariant sequence to an array of oligonucleotide probes immobilized on asolid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease, the sampleis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

[0075] Screening for mutations in PCLP-2 may be based on the functionalor antigenic characteristics of the protein. Protein truncation assaysare useful in detecting deletions that may affect the biologicalactivity of the protein. Various immunoassays designed to detectpolymorphisms in PCLP-2 proteins may be used in screening. Where manydiverse genetic mutations lead to a particular disease phenotype,functional protein assays have proven to be effective screening tools.The activity, e.g. selectin ligand functionality, of the encoded PCLP-2protein may be determined by comparison with the wild-type protein.

[0076] Diagnostic methods of the subject invention in which the level ofPCLP-2 expression is of interest will typically involve comparison ofthe PCLP-2 nucleic acid abundance of a sample of interest with that of acontrol value to determine any relative differences, where thedifference may be measured qualitatively and/or quantitatively, whichdifferences are then related to the presence or absence of an abnormalPCLP-2 expression pattern. A variety of different methods fordetermining the nucleic acid abundance in a sample are known to those ofskill in the art, where particular methods of interest include thosedescribed in: Pietu et al., Genome Res. (June 1996) 6: 492-503; Zhao etal., Gene (Apr. 24, 1995) 156: 207-213; Soares, Curr. Opin. Biotechnol.(October 1997) 8: 542-546; Raval, J. Pharmacol Toxicol Methods (November1994) 32: 125-127; Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216:299-304; Stolz & Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Honget al., Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem.(1984)143: 298. Also of interest are the methods disclosed in WO97/27317, the disclosure of which is herein incorporated by reference.

Screening Assays

[0077] The subject podocalyxin like proteins, e.g. PCLP and PCLP-2, finduse in various screening assays designed to identify therapeutic agents.The screening methods will typically be assays which provide forqualitative/quantitative measurements of the PCLP or PCLP-2 selectinligand and/or chemokine presenting activity in the presence of aparticular candidate therapeutic agent. For example, the assay could bean assay which measures the selectin ligand activity of PCLP or PCLP-2in the presence and absence of a candidate inhibitor agent. Thescreening method may be an in vitro or in vivo format, where bothformats are readily developed by those of skill in the art. Depending onthe particular method, one or more of, usually one of, the components ofthe screening assay may be labeled, where by labeled is meant that thecomponents comprise a detectable moiety, e.g. a fluorescent orradioactive tag, or a member of a signal producing system, e.g. biotinfor binding to an enzyme-streptavidin conjugate in which the enzyme iscapable of converting a substrate to a chromogenic product.

[0078] A variety of other reagents may be included in the screeningassay. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used.

[0079] A variety of different candidate agents may be screened by theabove methods. Candidate agents encompass numerous chemical classes,though typically they are organic molecules, preferably small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

[0080] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. PCLP-2 Nucleic Acid and Polypeptide TherapeuticCompositions

[0081] The nucleic acid compositions of the subject invention also finduse as therapeutic agents in situations where one wishes to enhancePCLP-2 activity in a host, e.g. in a mammalian host in which PCLP-2activity is sufficiently low such that a disease condition is present,etc. The PCLP-2 genes, gene fragments, or the encoded PCLP-2 protein orprotein fragments are useful in gene therapy to treat disordersassociated with PCLP-2 defects. Expression vectors may be used tointroduce the PCLP-2 gene into a cell. Such vectors generally haveconvenient restriction sites located near the promoter sequence toprovide for the insertion of nucleic acid sequences. Transcriptioncassettes may be prepared comprising a transcription initiation region,the target gene or fragment thereof, and a transcriptional terminationregion. The transcription cassettes may be introduced into a variety ofvectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and thelike, where the vectors are able to transiently or stably be maintainedin the cells, usually for a period of at least about one day, moreusually for a period of at least about several days to several weeks.

[0082] The gene or PCLP-2 protein may be introduced into tissues or hostcells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992),Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the DNA, then bombarded into skin cells.

Methods of Modulating PCLP or PCLP-2 Activity in a Host

[0083] Also provided are methods of regulating, e.g. inhibiting,podocalyxin like protein activity, e.g.. PCLP or PCLP-2 activity, suchas selectin ligand and/or chemokine presenting activity, in vivo in ahost. In such methods, an effective amount of active agent thatmodulates the selectin binding and/or chemokine presenting activity ofPCLP or PCLP-2 is administered to the host. In alternative embodiments,an agent that inhibits chemokine-PCLP-2 interaction is employed. Theactive agent may be a variety of different PCLP or PCLP-2 activitymodulators, where the modulator may act at a variety of different targetsites.

[0084] One type of active agent of interest is an agent that is capableof binding to PCLP or PCLP-2 in a manner such that the protein is nolonger capable of being recognized and/or bound by a selectin receptorand/or presenting chemokines. In yet other embodiments, the active agentmay be an agent that interacts with a target chemokine, e.g. SLC orMIP-3α, in a manner that inhibits chemokine-PCLP-2 interactions. Theactive agent may be a variety of different compounds, including anaturally occurring or synthetic small molecule compound, an antibody,fragment or derivative thereof, an antisense composition, and the like.

[0085] Naturally occurring or synthetic small molecule compounds ofinterest include numerous chemical classes, though typically they areorganic molecules, preferably small organic compounds having a molecularweight of more than 50 and less than about 2,500 daltons. Candidateagents comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, preferably atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

[0086] Active agents also include antibodies, both polyclonal andmonoclonal, and binding fragments thereof, such as Fv, F(ab′)₂ and Fab,where such antibodies preferably recognize a functional, e.g. the mucinor N-terminal, domain of PCLP-2. Such antibodies can be preparedaccording to methods known to those of skill in the art, where theantibodies may be humanized to improve host acceptance.

[0087] Suitable antibodies are obtained by immunizing a host animal withpeptides comprising all or a portion of a podocalyxin like protein ofthe subject invention, such as found in the PCLP or PCLP-2 polypeptidecompositions of the subject invention. Suitable host animals includemouse, rat sheep, goat, hamster, rabbit, etc. The origin of the proteinimmunogen may be mouse, human, rat, monkey etc. The host animal willgenerally be a different species than the immunogen, e.g. human PCLP-2used to immunize mice, etc.

[0088] The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part of thesubject protein, where these residues contain the post-translationmodifications, such as glycosylation, found on the native protein.Immunogens comprising the extracellular domain are produced in a varietyof ways known in the art, e.g. expression of cloned algenes usingconventional recombinant methods, isolation from HEC, etc.

[0089] For preparation of polyclonal antibodies, the first step isimmunization of the host animal with the subject protein, where thesubject protein will preferably be in substantially pure form,comprising less than about 1% contaminant. The immunogen may comprisecomplete podocalyxin like protein, fragments or derivatives thereof. Toincrease the immune response of the host animal, the subject protein maybe combined with an adjuvant, where suitable adjuvants include alum,dextran, sulfate, large polymeric anions, oil& water emulsions, e.g.Freund's adjuvant, Freund's complete adjuvant, and the like. The proteinmay also be conjugated to synthetic carrier proteins or syntheticantigens. A variety of hosts may be immunized to produce the polyclonalantibodies. Such hosts include rabbits, guinea pigs, rodents, e.g. mice,rats, sheep, goats, and the like. The protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

[0090] Monoclonal antibodies are produced by conventional techniques.Generally, he spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using PCLP or PCLP-2 bound to an insolublesupport, protein A sepharose, etc.

[0091] The antibody may be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequencesencoding the variable region of the heavy chain and the variable regionof the light chain are ligated to a spacer encoding at least about 4amino acids of small neutral amino acids, including glycine and/orserine. The protein encoded by this fusion allows assembly of afunctional variable region that retains the specificity and affinity ofthe original antibody.

[0092] For in vivo use, particularly for injection into humans, it isdesirable to decrease the antigenicity of the antibody. An immuneresponse of a recipient against the blocking agent will potentiallydecrease the period of time that the therapy is effective. Methods ofhumanizing antibodies are known in the art. The humanized antibody maybe the product of an animal having transgenic human immunoglobulinconstant region genes (see for example International Patent ApplicationsWO 90/10077 and WO 90/04036). Alternatively, the antibody of interestmay be engineered by recombinant DNA techniques to substitute the CH1,CH2, CH3, hinge domains, and/or the framework domain with thecorresponding human sequence (see WO 92/02190).

[0093] The use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987)J. Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

[0094] Antibody fragments, such as Fv, F(ab′)₂ and Fab may be preparedby cleavage of the intact protein, e.g. by protease or chemicalcleavage. Alternatively, a truncated gene is designed. For example, achimeric gene encoding a portion of the F(ab′)₂ fragment would includeDNA sequences encoding the CHl domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

[0095] Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

[0096] Expression vectors include plasmids, retroviruses, YACs, EBVderived episomes, and the like. A convenient vector is one that encodesa functionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

[0097] Particular antibodies of interest for inhibiting PCLP or PCLP-2activity include: MECA-79, 2H5 as described in Sawada et al., BiochemBiophys. Res. Com. 193: 337-347; and the like.

[0098] Modulators of interest as active agents also include agents thatalter the structure, usually by chemical alteration through thedisruption of one or more covalent bonds, of the PCLP or PCLP-2,particularly the mucin domain, in a manner such that the protein is nolonger capable of being bound by a selectin receptor. Such agentsinclude enzymes which cleave one or more bonds present in the proteinstructure and thereby disrupt the structure such that it is no longerrecognized by a selectin receptor. Such enzymes include: endopeptidases,such as O-sialoglycoprotein endopeptidase, and the like; sialidases,such as Arthrobacter ureafaciens sialidase, Clostridium perfringenssialidase, New Castle Disease Virus sialidase, and the like; sulfatases,such as Gal-6-sulfatase and GlcNAc-6-sulfatase and the like; proteases,such as OSGE, catalyitic antibodies, and the like; fucosidases; and thelike

[0099] In yet other embodiments of the invention, the active agent is anagent that modulates, and generally decreases, the expression of theprotein, e.g. PCLP or PCLP-2. Such agents include antisense reagents,e.g. DNA or RNA, expression repressing agents, and the like. Theanti-sense reagent may be antisense oligonucleotides (ODN), particularlysynthetic ODN having chemical modifications from native nucleic acids,or nucleic acid constructs that express such anti-sense molecules asRNA. The antisense sequence is complementary to the mRNA of the targetedgene, and inhibits expression of the targeted gene products. Antisensemolecules inhibit gene expression through various mechanisms, e.g. byreducing the amount of mRNA available for translation, throughactivation of RNAse H, or steric hindrance. One or a combination ofantisense molecules may be administered, where a combination maycomprise multiple different sequences.

[0100] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucieotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

[0101] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0102] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993), supra, and Milliganet al., supra.) Preferred oligonucleotides are chemically modified fromthe native phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

[0103] Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3′-O-′-5′-S-phosphorothioate, 3′-S-5′O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5- propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

[0104] As an alternative to anti-sense inhibitors, catalytic nucleicacid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be usedto inhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the host, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434-42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a met al. complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56.

[0105] In yet other embodiments, the agent is an agent thatpost-translationally alters the protein, e.g. PCLP or PCLP-2, in amanner such that an active protein is not presented on the cell surface.Such agents include: inhibitors of glycosyl or sulfotransferases,,andthe like.

[0106] In yet other embodiments, the active agent will be a podocalyxinlike protein competitor, e.g. a PCLP or PCLP-2 competitor, where suchcompetitors include the selectin ligands of the subject invention,described above, including soluble PCLP or PCLP-2 and selectin bindingfragments thereof, which may be derived from natural sources orsynthetically produced, e.g. by recombinant DNA technology such asexpression of a recombinant gene encoding PCLP or PCLP-2 in anenvironment capable of post-translationally modifying the expressedrecombinant protein to obtain compound with PCLP or PCLP-2 like selectinbinding activity.

[0107] As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. Generally, the desired result isat least a reduction in the amount of selectin binding as compared to acontrol.

[0108] In the subject methods, the active agent(s) may be administeredto the host using any convenient means capable of resulting in thedesired inhibition of PCLP and/or PCLP-2 activity, e.g. selectin bindingactivity, chemokine presenting activity, etc. Thus, the agent can beincorporated into a variety of formulations for therapeuticadministration. More particularly, the agents of the present inventioncan be formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols.

[0109] As such, administration of the agents can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

[0110] In pharmaceutical dosage forms, the agents may be administered inthe form of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

[0111] For oral preparations, the agents can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0112] The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

[0113] The agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0114] Furthermore, the agents can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The compounds of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

[0115] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

[0116] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0117] The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0118] Those of skill will readily appreciate that dose levels can varyas a function of the specific compound, the severity of the symptoms andthe susceptibility of the subject to side effects. Preferred dosages fora given compound are readily determinable by those of skill in the artby a variety of means.

[0119] The subject methods find use in the treatment of a variety ofdifferent disease conditions involving selectin-podocalyxin likeprotein, e.g. selectin-PCLP or selectin- PCLP-2, binding interactionsand/or chemokine-leukocyte interactions, particularly L-,E- or P-selectin mediated binding events. Such disease conditions include thosedisease conditions associated with or resulting from the homing ofleukocytes to sites of inflammation, the normal homing of lymphocytes tosecondary lymph organs, the interaction of platelets with activatedendothelium, platelet-platelet and platelet-leukocyte interactions inthe blood vascular compartment; and the like. Accordingly, specificdisease conditions that may be treated with the subject methods include:acute or chronic inflammation; autoimmune and related disorders, e.g.systemic lupus erythematosus, rheumatoid arthritis, polyarteritisnodosa, polymyositis and dermatomyositis, progressive systemic sclerosis(diffluse scleroderma), glomerulonephritis, myasthenia gravis, Sjogren'ssyndrome, Hashimoto's disease and Graves' disease, adrenalitis,hypoparathyroidism, and associated diseases; pernicious anemia;diabetes; multiple sclerosis and related demyelinating diseases; uveitispemphigus and pemphigoid; cirrhosis and other diseases of the liver;ulcerative colitis; myocarditis; regional enteritis; adult respiratorydistress syndrome; local manifestations of drug reactions (dermatitis,etc.); inflammation-associated or allergic reaction patterns of theskin; atopic dermatitis and infantile eczema; contact dermatitis,psoriasis lichen planus; allergic enteropathies; atopic diseases, e.g.allergic rhinitis and bronchial asthma; transplant rejection (heart,kidney, lung, liver, pancreatic islet cell, others); hypersensitivity ordestructive responses to infectious agents; poststreptococcal diseasese.g. cardiac manifestations of rheumatic fever, etc.; tissue rejectionduring transplantation; atherosclerosis; restenosis after angioplasty;damaging thrombotic events, reperfusion injury; and the like.

[0120] By treatment is meant at least an amelioration of the symptomsassociated with he pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as inflammation and painassociated therewith. As such, treatment also includes situations wherethe pathological condition, or at least symptoms associated therewith,are completely inhibited, e.g. prevented from happening, or stopped,e.g. terminated, such that the host no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

[0121] A variety of hosts are treatable according to the subjectmethods. Generally such hosts are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the hosts will behumans.

[0122] Kits with unit doses of the active agent, usually in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the drugs intreating pathological condition of interest. Preferred compounds andunit doses are those described herein above.

[0123] The following examples are offered primarily for purposes ofillustration. It will be readily apparent to those skilled in the artthat the formulations, dosages, methods of administration, and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

Experimental

[0124] I. PCLP

[0125] A. Materials and Methods

[0126] 1. Antibodies carbohydrates and Ig-chimeras

[0127] Mouse anti-PCLP monoclonal antibodies. 3D3(IgG) 2A4 (IgM), and4F10 (IgM) were generated as described in Kershaw et al., J. Cell Biol.(1997)272:15707-15714 and produced as hybridoma culture supernatants.Additionally, the 3D3 antibody was produced as ascites. The MECA-79 (RatIgM) hybridoma was obtained from the American Type Culture Collection(Rockville, Md.), produced as ascites and purified on Sepharose-coupledanti-rat IgK (Zymed, South San Francisco, Calif.) as recommended by themanufacturer. Antibodies were biotinylated with NHS-biotin (Sigma, St.Louis Mo.) as described in Harlow & Lane, Antibodies: A LaboratoryManual, (Cold Spring Harbor Press,1988). Purified anti-L-selectinmonoclonal antibody LAM1-3 was obtained from Dr. Thomas Tedder (DukeUniversity, NC). Fucoidin was purchased from Sigma (St. Louis, Mo.).L-selectin/IgG and CD4/IgG chimeras consist of the entire extracellulardomain of murine L-selectin or human CD4 fused to the Fc domain of humanIgG1. These chimeric proteins were collected from the supernatants oftransfected 293 cells and protein-A purified as described previously(Watson et al., J. Cell Biol. (1990)117:895). Arthrobacter urafacienssialidase was purchased from Oxford glycosystems (Oxford, UK).O-sialoglycoprotein endopeptidase (OSGE) was purchased from Cedar LaneBiologicals (Ontario, Canada).

[0128] B. Cells

[0129] Jurkat T cells were obtained from the laboratory of Dr. Art Weiss(University of California, San Francisco) and were maintained inRPMI-1640 medium supplemented with 100 U/ml penicillin, 100 μg/mlstreptomycin, 2 mM glutamine and 5% heat inactivated FCS (Hyclone,Logan, Utah). Human T-lymphocytes were isolated from venous bloodsamples using standard gradient techniques as described in Giblin etal., J. Immunol. (1997) 159:3498-3507. Briefly, blood was collected fromhealthy donors and EDTA was added to a final concentration of 5 mM toprevent clotting and cell activation. The blood samples were dilutedwith ½ volume of cation-free HBSS and layered over Histopaque 1077(Sigma). After centrifugation at 500×g for 20 minutes at roomtemperature, the peripheral blood mononuclear cell (PBMC) fraction wascollected, diluted in cold cation-free HBSS supplemented with 0.2% BSAand centrifuged to recover the isolated cells. T-lymphocytes wereobtained by depletion of B cells and monocytes. PBMC were treated withsaturating amounts of mouse anti-CD14 (monocyte marker), mouse anti-CD19(B cell marker) followed by sheep anti-mouse IgG-coupled paramagneticparticles (Dynal, Lake Success, N.Y.). Labeled cells were then removedusing a magnet (Dynal).

[0130] C. Immunohistochemistry

[0131] Surgical specimens of human tonsil (obtained from the Universityof California, San Francisco) were either embedded in O.C.T. media(Miles Inc., Elkhart Ind.) and frozen, or fixed in neutral bufferedformalin and parafin embedded. Sections of paraffin-embedded human lymphnode or appendix were obtained from archived specimens that had beendetermined to be devoid of specific pathology. 12 μm frozen sectionswere fixed in 1% paraformaldehyde in 50mM cacodylate, pH 7 for 20 min.For horseradish peroxidase detection, endogenous peroxidase was quenchedwith 0.3% H₂O_(2,) 0.2 M NaN₃ in PBS, for 20 min. Sections were blockedwith 5% goat serum and stained with undiluted hybribdoma culturesupernatant overnight at 4° C. The sections were then incubatedsequentially with 1:250 diluted biotinylated goat anti- mouse IgG (heavyand light chain specific) (Vector, Burlingame, Calif.) in 5% goat serumand 2.5% human serum, then with ABC elite reagent (Vector) in PBS, andfinally, with AEC chromagen (Biomeda, Foster City, Calif.). Afterchromagen development, the sections were counterstained with aqueoushematoxylin and mounted in Permafluor (Lipshaw, Pittsburgh, Pa.). PBSwas used to wash in after each incubation. For fluorescent detection,frozen sections were fixed, blocked and incubated with PCLP antibodiesas above. Sections were then incubated sequentially with FITC-conjugatedgoat anti-mouse IgG (Zymed, South San Francisco, Calif.) in 5% goatserum and 2.5% human serum; 1 ug/ml biotinylated MECA-79 in 5% goatserum, 5% human serum, and 2.5% rat serum;and Texas red conjugatedstreptavadin Vector) in 5% goat serum, 2.5% human serum, and 2.5% ratserum. The sections ere washed in PBS after each step and mounted invectasheild (Vector).

[0132] D. PNAd Isolation

[0133] Frozen surgical specimens of human tonsils were obtained from theCooperative Human Tissue Network, Western Division, (Case WesternUniversity) which is funded by the National Cancer Institute. The tissuewas homogenized in PBS containing 2% Triton X-100, 1 μg/ml leupeptin, 10μg/ml aprotinin, 1 μM pepstatin, 10 μg/ml Pefablock, and 5 mM EDTA in aglass/teflon homogenizer. This lysate was rocked at 4° C. for 30 min.followed by centrifugation at 20,000 ×g for 20 min. at 4° C. The lysatewas then diluted 2-fold with PBS and passed through a 0.2 μm filter.This clarified lysate was passed over a 1 ml Sepharose-4B column (CNBractivated, Sigma) that had been coupled to 2.4mg of affinity purifiedMECA-79. The column was washed with PBS containing 0.1% Triton X-100,PBS-TX, followed by PBS containing 25mM n-octylglucoside. PNAd was theneluted with 100mM triethylamine containing 25mM n-octylglucoside andneutralized with {fraction (1/10)} volume 3M Tris pH 6.8. This materialwas concentrated using a centricon-30 microconcentrator, diluted withPBS containing 25mM n-octylglucoside and reconcentrated. The purifiedprotein was quantified using the BCA assay (Pierce, Rockford, Ill.), andpurity was assessed by SDS-PAGE followed by silver-enhanced alcian bluestaining based on previously described procedures (See Moller et al.,Anal. Biochem. (1993)209:169-175. 7.5% SDS-PAGE gels were fixed in 25%ethanol, 10% acetic acid overnight. They were then stained with 0.125%alcian blue in fix solution for 1 hr. followed by three, 30 mindestaining steps with fix solution. Gels were then incubated in 5%gluteraldehyde in H₂O for 1 hr. followed by 4, 30 min. washes with 10%ethanol, 5% acetic acid. Gels were then washed 3 times with water for 10min. each and incubated for 30 min. in 0.4% silver nitrate. Gels werethen rinsed briefly in water and developed with 0.013% formaldehyde in2.5% sodium carbonate. The development was stopped with 1% acetic acid.

[0134] E. Immunoprecipitation and Western Blotting

[0135] Protein A-Sepharose Fast Flow beads (Pharmacia, Uppsala, Sweden)were loaded with 200 μl of 3D3 hybridoma culture supernatant or 6 μgpurified mouse IgG (Caltag, San Francisco, Calif.) and washed withPBS-TX. 100 μM of PBS-TX containing approximately 50 ng PNAd and 0.1 μlNGS was then added to the beads and rocked overnight at 4° C. Thesupernatant was collected and pooled with two 100 μl PBS-TX washes ofthe beads. This unbound fraction was then precipitated with 4 volumes ofacetone at -−20° C for 1 hr. collected by centrifugation, dissolved inSDS-PAGE sample buffer and boiled. The beads were washed extensivelywith PBS-TX and eluted by boiling in SDS-PAGE sample buffer. 7.5%SDS-PAGE gels were transferred in a Hoeffer TE series electroblotter toProblott (Applied Biosystems, Foster City, Calif.) in 10 mM CAPS, 10%methanol at 22 V for 16 hours. The blot was then blocked in PBScontaining 0.1% Tween-20, PBST, and 3% nonfat dry milk for 1 hr. Blotswere then sequentially incubated in either lug/ml MECA-79 or {fraction(1/50)} diluted 3D3 supernatant in blocking solution, {fraction(1/1000)} diluted biotinylated mouse anti-rat Igμ (Caltag, South SanFrancisco, Calif.) or {fraction (1/1000)} diluted biotinylated goatanti-mouse IgG (Vector) in blocking solution, and finally {fraction(1/2000)} diluted horseradish peroxidase-conjugated streptavadin(Caltag) in PBST. Blots were washed with PBST after each incubation anddeveloped using an ECL substrate (Amersham, Arlington Heights, Ill.).For detection with the 3D3 mAb, 5-10 times more PNAd was used than forMECA-79 detection.

[0136] F. Detection of PCLP mRNA

[0137] High endothelial cells (HEC) were purified from surgicalspecimens of human tonsils by immunomagnetic selection with MECA-79using a modification of a previously described procedure (Girard &Springer, Immunology Today (1995) 16: 449-457. After collagenasedigestion of the tonsils, stromal cells were resuspended at 2×10⁸cells/ml in staining buffer (PBS containing 1% BSA and 5mM EDTA) andincubated with 0.3 μg of MECA-79 per 10⁷ cells on ice for 15 min. Cellswere collected by centrifugation, washed once in staining buffer andincubated with 30 μl of biotinylated mouse anti-rat Igμ (Caltag) for 15min. on ice. Cells were washed with staining buffer again and 300 μl ofstreptavadin coupled paramagnetic beads (Miltani, Auburn, Calif.) wereadded and incubated on ice for 15 min. MECA-79 positive cells wereisolated using an MS+column and a “mini-MACS” magnet (Miltani) asrecommended by the manufacturer. Purity was assessed microscopically bymorphologic criteria as well as MECA-79 expression by immunofluorescenceusing cyanine-3-conjugated streptavadin (Caltag).

[0138] Human umbilical vein endothelial cells, HUVEC, were purchasedfrom Clonetics (San Diego, Calif.) and grown as recommended. Tonsillarlymphocytes were prepared by mincing surgical specimens of human tonsiland flushing the loose lymphocytes through a fine screen with coldRPMI-1640 media.

[0139] Total RNA was isolated from HEC, HUVEC and lymphocytes usingRNAzol B (Tel-Test, Friendswood, Tex.). First strand cDNA was made from2 μg total RNA primed with random hexamers using AMV-reversetranscriptase (Gibco-BRL) according to the manufacturer'srecommendations. Serial dilutions of cDNA were amplified using Klentaqpolymerase mix (Clontech, Palo Alto, Calif.) and the following primers:for PCLP,

[0140] 5′-TTTGGATCCCAGATGCCAGCCAGCTCTACG-3′ and

[0141] 5′-TTTGAATTCCTTCATGTCACTGACCCCTGC-3′ were used, and forhypoxanthine phosphoribosyltransferase, HPRT,

[0142] 5′-CTGCTGGATTACATCAAAGCACTG-3′ and

[0143] 5′-TCCAACACTTCGTGGGGTCCT-3′ were used. One half of the resultingamplified DNA was electrophoresed on 1% agarose gels and visualized byethidium bromide staining.

[0144] G. Purification of MECA-79 reactive PCLP

[0145] A detergent lysate of was made from 10 g of human tonsils asdescribed for PNAd isolation. This material was passed twice over a 2 mlcolumn of protein-A coupled Sepharose. The unbound material was thenpassed over a 0.5 ml protein-A column to which 2 ml of 3D3 ascites hadbeen bound and covalently coupled with dimethylpamylidate (Pierce,Rockford, Ill.) as described (Harlow & Lane, 1988). After washing withPBS-TX, the bound material was eluted with 100mM triethylamine with 0.1%Triton X-100 and neutralized with {fraction (1/10)} vol 3M Tris, pH6.8.This sample was then purified on MECA-79 coupled Sepharose as describedfor PNAd isolation. {fraction (1/60)} of the resulting material wasanalyzed by SDS-PAGE followed by MECA-79 western blotting.

[0146] H. Parallel Plate Flow Chamber Analysis

[0147] Purified PCLP was diluted 1:10 or 1:20 in Tris-buffered saline(TBS) pH8.5 and coated onto bacteriological Petri dishes (Corning, SanMateo, Calif.) at 4° C. overnight. For blocking, the plates were treatedwith 3% BSA for one hour at room temperature. The substrate-coatedslides were incorporated as the lower wall in a parallel plate flowchamber and mounted on the stage of an inverted phase-contrastmicroscope (Diaphot 200; Nikon Inc., Garden City, N.Y.). All flowexperiments were performed at room temperature. The wall shear stresswas calculated as previously described in Lawrence & Springer, Cell(1991) 65:859-873. Cells were stored at a concentration of 1×10⁷cells/ml in cation-free HBSS containing 0.2% BSA and were diluted 1:10in HBSS containing 0.2% BSA immediately before perfusion into the flowchamber. Jurkat cells were infused into the chamber at a shear stress of0.8 dynes/cm² and T-lymphocytes at 1.25 dynes/cm². After three minutes,the cell tethering rate (number of cells that tethered per minute perfield) was determined by analysis of two to four fields of view. Forinhibition studies, the cells (10⁷/ml) were preincubated for 20 min incation-free HBSS containing 0.2% BSA with 150 μg/ml LAM1-3(anti-L-selectin) or 100 μg/ml fucoidin (Sigma). The cell suspension wasdiluted 10-fold with HBSS containing 0.2% BSA before infuision into theflow chamber. The substrate was incubated with MECA-79 (ascites diluted1:20) or 3D3 (anti-PCLP, ascites diluted 1:20) for one hour at roomtemperature. Coated PCLP was treated with 50 mU/ml of sialidase in 0.1 Msodium acetate pH 5.0 or with 0.1 M sodium acetate pH 5.0 alone as acontrol. O-sialoglycoprotease was diluted 10-fold in PBS to aconcentration of 0.24 mg/ml total protein and used to treat immobilizedPCLP for one hour at room temperature.

[0148] B. Results

[0149] 1. High Endothelial Cells Express PCLP

[0150] Three different monoclonal antibodies specific for human PCLPwere used for immunohistochemical staining of sections of humansecondary lymphoid organs. First, two color immunofluorescenceexperiments were performed by simultaneously staining frozen sections ofhuman tonsil with the PCLP mAb, 4F10, and MECA-79 HEV were observed thatexpressed both PCLP and the MECA-79 epitope. PCLP was concentrated atthe surface of the high endothelial cells on the luminal aspect of thevessel. Similar staining patterns were obtained with two other PCLPmAbs, 3D3 and 2A4. Localization to the luminal face of vascularendothelium has also been reported for podocalyxin in kidney (Keijaschkiet al., J. Cell Biol. (1984) 98:1591-1596) and for thrombomucin inmultiple tissues (McMagny et al., J. Cell Biol. (1997) 138: 1395-1407.HEV of paraffin-embedded samples of lymph node and appendix were alsostained by the PCLP mAb, 2A4, demonstrating that PCLP expression is acharacteristic of HEV in both mucosal and non-mucosal lymphoid organs.

[0151] PCLP expression was not restricted to HEV, as staining wasassociated with endothelial cells of many classes of blood vessels inthese lymphoid organs. PCLP staining was also found in musculararteries, arterioles and capillaries. These findings are in agreementwith previous studies demonstrating expression of podocalyxin and itshomologs on vascular endothelium in non-lymphoid tissues. See Kershaw etal., supra; McMagny et al., supra and Keijaschki et al., supra.

[0152] It was also apparent from these experiments that while themajority HEV in tonsils were positive for PCLP expression, a minoritywere weak or negative. This observation was also noted in the two colorimmunofluorescent experiments, where some MECA-79 positive vesselsexpressed little or no PCLP. In contrast, CD34 can be detected onvirtually all HEV in sections of the same tonsil. These PCLP-negativeHEV were, however, not noted in lymph node or appendix, suggesting thatthis heterogeneity was restricted to tonsil and was possibly the resultof the inflamed state of the tissue.

[0153] A RT-PCR assay was then used to demonstrate the presence of PCLPmRNA in purified high endothelial cells (HEC). MECA-79 expressing cellswere purified from collagenase-digested tonsillar stroma byimmunomagnetic selection. This procedure produces greater than 95% pureHEC with a slight contamination of lymphocytes as assessed by morphologyand MECA-79 expression. Total RNA was extracted from HEC, tonsillarlymphocytes, and primary cultured endothelial cells, HUVEC. Serialdilutions of reverse transcribed cDNA was then amplified with primersspecific for PCLP or hypoxanthine phosphoribosyltransferase (HPRT). Thelevel of the constitutively expresed HPRT mRNA was used to normalize theamount of total cDNA in each sample. PCLP mRNA was readily detected inboth HEC and HUVEC cDNA, but was only barely detectable in thelymphocyte sample. When the data was normalized for HPRT expression,PCLP mRNA levels were approximately two-fold greater in HUVEC than inHEC. Importantly, the PCLP expression level in HEC was 16 times higherthan in the lymphocyte sample, establishing that the PCLP mRNA detectedin the HEC preparation was not a result of lymphocyte contamination.Additionally, when reverse transcriptase was omitted from the cDNAreactions, no PCR product was observed, demonstrating that all productswere the result of amplified cDNA and not contaminating genomic DNA.

[0154] 2. Tonsillar PCLP displays the MECA-79 epitope and bindsL-selectin

[0155] In order to determine whether HEV derived-PCLP carries theMECA-79 epitope and is therefore a potential ligand for L-selectin, wesubjected MECA-79 purified tonsil lysate (i.e. PNAd) to western blottingwith the PCLP mAb 3D3 as well as MECA-79 (FIG. 4). MECA-79 detectedmajor bands of Mr 210, 160, 115, and 60, in agreement with previouscharacterizations (See Berg et al., J. Cell Biol. (1991) 114: 343-349.The 3D3 antibody specifically recognized a Mr 160 component in PNAdsuggesting that this band represented PCLP.

[0156] The identity of the Mr 160 component of PNAd as PCLP wasconfirmed by immunoprecipitation with the 3D3 antibody. When PNAd wasimmunoprecipitated with 3D3, the Mr 160 band was specifically depletedfrom the unbound fraction, and only this component was found in thebound fraction. This result demonstrates that the Mr 160 bandrepresented a MECA-79 reactive form of PCLP. In multiple experiments,the 3D3 antibody reproducibly depleted a maximim of 50% of the Mr 160band. Increasing the amount of antibody did not enhance theprecipitation, suggesting that there were glycoforms of HEV-derived PCLPwhich were unreactive with the 3D3 antibody. Since this antibody wasmade against a non-glycosylated form of PCLP that had been produced inE. coli, the existence of a native glycosylated molecule that isunreactive would not be surprising. It is, however, formally possiblethat the unreactive material represented a distinct glycoprotein withthe same apparent molecular weight.

[0157] In order to demonstrate a direct interaction between HEV-derivedPCLP and L-selectin, the same preparation of PNAd was subjected toprecipitation with a recombinant L-selectin/IgG chimera. Essentially allof the PCLP band and more that 90% of the PNAd was precipitated with theL-selectin/IgG chimera, whereas no interaction was observed between PNAdand a control CD4/IgG chimera. Furthermore, the bound material could beeluted by the addition of EDTA, demonstrating that PCLP and CD34 as wellas the two unidentified PNAd components (Mr 200 and 65) bound toL-selectin in a calcium-dependent manner, which is characteristic ofselectin-carbohydrate interactions (See Rosen & Bertozzi, CurrentBiology (1994) 6:663-673.

[0158] 3. HEV-derived PCLP is a sialomucin

[0159] PNAd was subjected to digestion with O-sialoglycoproteinendopeptidase, OSGE, a metalloprotease which specifically hydrolyzes thepeptide backbone of sialomucins (See Mellors A. & Lo, Methods inEnzymology (1995)248:728-740. When PNAd was treated with OSGE, virtuallyall of the material detected by MECA-79 was eliminated, including the Mr160 PCLP band. Interestingly, no low molecular weight cleavage productswere detected, suggesting that either the resulting cleaved productswere very small or that an intact peptide backbone is essential for thepresentation of the MECA-79 epitope.

[0160] In order to directly demonstrate the sialylation of HEV-derivedPCLP, we subjected PNAd to sialidase digestion followed by westernblotting with the 3D3 antibody. This treatment resulted in a clearincrease in the electrophoretic mobility of PCLP demonstrating thepresence of sialic acid. Although removal of sialic acid from manyglycoproteins including glomerular PCLP results in a decrease inmobility due to a loss of charge, PCLP's behavior was similar to that ofHEV-derived GlyCAM-1, and CD34, as well as the other PNAd components(data not shown). From the above, it can be concluded that all PNAdcomponents are sulfated since MECA-79 has been shown to bind asulfate-dependent epitope. The unusual electrophoretic behavior of PCLPafter sialidase treatment was therefore attributed to the presence ofsulfate which maintains the negative charge of the glycoprotein afterthe loss of sialic acid. The sensitivity of PCLP to both OSGE andsialidase demonstrates that its HEV-derived form is sialomucin-like instructure as are CD34 and all other known L-selectin ligands in HEV.

[0161] Western blotting of PNAd with the 3D3 antibody often revealed ahigh molecular weight band (>Mr 200) in addition to the major Mr 160band. This band was suspected to the SDS-stable multimerized form ofPCLP which has been previously described in Dekan et al., 1991). Themolecular weight of this band did not correspond to the major Mr 210component of PNAd. However, a minor band with a similar molecular weightto this putative multimer was occasionally observed in PNAd and likelyrepresents the same species.

[0162] 4. PCLP mediates L-selectin dependent adhesion under flow

[0163] MECA-79 reactive PCLP was purified from tonsil lysates bysequential purification on 3D3 coupled Sepharose followed by MECA-79coupled Sepharose. Western blotting of the purified material withMECA-79 showed a major band of Mr 160 as expected. This material wascoated onto one surface of a parallel plate laminar flow chamber andeither peripheral blood T-lymphocytes or Jurkat T-lymphoma cells wereinfused into the chamber at physiological flow rates. Under theseconditions, both cell types transiently adhered to the PCLP-coatedsurface, but not to a surface coated with bovine serum albumin. At 0.8dynes/cm² the velocity of Jurkat cells rolling on PCLP was 50.1+/−14.1μm/sec, which was comparable to the velocity of these cells rolling onunfractionated PNAd (data not shown). This adhesion was dependent onL-selectin, since it was completely inhibited by pretreatment of theJurkat cells with either an L-selectin mAb or the L-selectin antagonistfucoidin. Furthermore, treatment of the coated PCLP with eitherO-sialoglycoprotein endopeptidase or Arthobacter urefaciens sialidaseabrogated the interaction with Jurkat cells, demonstrating thesialomucin-like structure of PCLP and the absolute requirement of sialicacid for L-selectin binding. Finally, treatment of the coated substratewith MECA-79 abrogated all interaction with Jurkat cells, establishingthat lymphocyte L-selectin in was binding to the immobilized PCLP andnot to a MECA-79 unreactive contaminant. In contrast, treatment of theimmobilized PCLP with the 3D3 antibody did not affect lymphocyteadhesion. This result was not suprising since this antibody recognizes aprotein determinant. Based on these data, we conclude that immobilized,MECA-79 reactive PCLP is able to mediate the L-selectin-dependenttethering and rolling of lymphocytes under physiological flowconditions.

[0164] II. PCLP-2

[0165] A. Identification and cloning of PCLP-2

[0166] Alignment of the cytoplasmic tails of CD34 and PCLP revealssignificant sequence homology. By querying the NCBI Gene Bank EST database using the cytoplasmic tail of PCLP, we were able to identifyseveral mouse EST's which represent the same gene and share considerablehomology to PCLP and CD34. One such EST clone was obtained and sequencedto verify its identity. Using this clone as a probe, northern blots wereperformed on mRNA from various mouse tissues to determine the size ofthe transcript and its expression pattern. A single 2.5 kb transcriptwas detected which was abundantly expressed in the brain and to a lesserextent in heart, testes, kidney, and liver. In order to clone thefull-length human cDNA clone, PCR primers were generated based on thismouse sequence which amplified a specific product from both mouse andhuman cDNA. A PCR assay utilizing these primers was used to screen ahuman brain library, which was obtained as 96 master pools of 5000plasmid clones each. Subpools containing 100 E. coli clones each wereobtained from several positive master pools. Positive subpools werespread on LB plates and individual colonies were screened to obtainsingle positive clones. This strategy resulted in the isolation of twoindependent clones, the longest of which was 2.3kb in length (see FIG.1). This cDNA (SEQ ID NO:01) predicts a protein of 605 amino acids (SEQID NO:02) which contains an approximately 170 amino acid N-terminaldomain consisting of 30% acidic residues as well as two tyrosines whichmay be sulfated. This acidic domain is followed by a 140 amino acid longmucin-like domain (35% serine, threonine and proline), acysteine-containing domain, a predicted transmembrane segment, and an 80amino acid long cytoplasmic tail. Other predicted features include 5potential glycosaminoglycan (GAG) attachment sites (one in theN-terminal acidic domain), and three potential N-linked glycosylationsites. Expression of bases 1-630 of this cDNA, representing theamino-terminal acidic domain, fused to the Fc domain of human IgGl inCOS-7 cells produced a major protein band of about 70 kD which waspurified on protein A-sepharose and subjected to amino-terminal sequenceanalysis. Glycine-33 was identified as the amino-terminus of the matureprotein indicating that the preceding amino acids represent a cleavablesignal peptide.

[0167] B. Production of Antibodies to PCLP-2

[0168] Two domains of PCLP-2 were expressed in E. coli as 6-His fusionproteins, purified and used to immunize rabbits. The membrane-proximalcysteine-containing domain and the cytoplasmic tail were chosen asimmunogens. Both antisera react with the immunizing antigen by ELISA andwestern blot. In order to improve specificity, antibodies from both serawere affinity purified on antigen-coupled sepharose.

[0169] C. Expression Pattern of Human PCLP-2

[0170] Using the human PCLP-2 clone as a probe, northern blots wereperformed to determine the expression pattern of this gene in humantissues. As seen in mouse, a 2.5kb transcript was detected predominantlyin brain, while lower expression was noted in pancreas, kidney, muscle,adult and fetal liver, bone marrow, peripheral blood leukocytes, thymus,lymph node, and spleen. To show that, like CD34 and PCLP, PCLP-2 wasexpressed by endothelial cells, RT-PCR was used to detect PCLP-2 mRNA incDNA derived from cultured human umbilical vein endothelial cells(HUVEC). PCLP-2 mRNA was also detected in HEC purified from humantonsils by immunomagnetic selection using the MECA-79 antibody.Endothelial expression of PCLP-2 was confirmed by staining frozensections of human tonsils with the anti- PCLP-2 antibodies describedabove. Staining was seen only in the vascular endothelium including HEV.

[0171] D. Biochemical Characterization of PCLP-2

[0172] The full-length PCLP-2 cDNA was expressed in COS-7 cellsmetabolically labeled with ³⁵SO₄ and cell lysates were analyzed bySDS-PAGE. Transfection of PCLP-2 but not CD34 resulted in the productionof a 200 kD sulfated protein. This 200kD band was shown to representPCLP-2 by immunoprecipitation with specific antibodies. Since thepredicted molecular weight of the PCLP-2 peptide is 65kD, we suspectthat the native molecule is extensively modified with carbohydrate. Thestrong incorporation of ³⁵SO₄ (much more than CD34) lead us to suspectthe presence on sulfated GAG chains and/or the sulfation of the twoextracellular tyrosines.

[0173] In order to produce more material for biochemicalcharacterization, the extracellular domain of PCLP-2 was fused to the Fcdomain of human IgGl (PCLP-2/IgG) in order to create a soluble, secretedprotein. At the same time, an IgG fusion protein was made whichcontained only the N-terminal acidic domain of PCLP-2 (N- Term/IgG).Expression and ³⁵SO₄ labeling of these PCLP-2 constructs, but notexpression of the IgG Fc domain alone resulted in secreted, sulfatedmolecules which could be purified by precipitation with anti-humanIgG-agarose. The presence of chondroitin sulfate on both PCLP-2constructs was demonstrated by digestion of these fusion proteins withGAG degrading enzymes. A mixture of Heparinases had no effect, butchondroitinase ABC removed the majority of the sulfate from both PCLP-2fusion proteins. Notably, a low molecular weight species was unaffectedby chondroitinase in both cases. This species likely contains anothersulfate modification, of either carbohydrate or tyrosine. In order todemonstrate the mucin-like character of PCLP-2, the fusion proteins weresubjected to digestion with O-sialoglycoprotein endopeptidase (OSGE).Only the full-length fusion protein, which contains the mucin-likedomain, was degraded by OSGE, demonstrating the mucin-like character ofthis domain.

[0174] E. PCLP-2 Binds Chemokines

[0175] Many chemokines are known to contain a basic heparin-bindingdomain. Because of the acidic nature of the N-terminal domain of PCLP-2,we investigated whether chemokines could bind this protein. This woulddefine a potential novel mechanism of chemokine immobilization on theendothelial surface. An ELISA assay was developed in which PCT P-2/IgGfusion proteins are immobilized, and the binding of FLAG-tagged SLC canbe detected with a biotinylated anti-FLAG antibody. Using this assay, wehave shown that SLC/FLAG binds to PCLP-2/IgG, but not to CD34/IgG. SinceCD34 and PCLP-2 are structurally similar except for the acidicN-terminus of PCLP-2, we suspected that SLC was binding to this uniquedomain. To demonstrate this, the N-Term/IgG fusion protein wasimmobilized and shown to support a similar level of binding of SLC asthe full-length protein. Specificity of binding was demonstrated byinhibition with either heparin or with SLC/FLAG. In order to determineif other chemokines could bind to PCLP-2, assays were performed in whichunlabeled chemokines were tested for their ability to block the bindingof N- Term/IgG to SLC/FLAG. Of the small panel tested thus far, MIP3αwas able to efficiently inhibit SLC/FLAG binding to PCLP-2, but SDF-1αand ELC were not. These results demonstrate that some, but not allchemokines are able to bind to the amino-terminal domain of PCLP-2.

[0176] It is evident from the above results and discussion that PCLP andPCLP-2 are glycoprotein members of the PNAd family of glycoproteins. Itis also evident that new methods of inhibiting selectin binding eventsand/or leukocyte emigration are provided. As such, the subject inventionprovides for additional means to elucidate the mechanics underlying celltrafficking. In addition, the subject invention provides new means fortreating disease conditions resulting from selectin binding events andor leukocyte emigration, such as acute and chronic inflammation, and thelike.

[0177] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0178] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1 2 1 2269 DNA human 1 ccagttccgg cacgaggacc atgggccggc tgctgcgggccgcccggctg ccgccgctgc 60 tttcgccgct gctgcttctg ctggttgggg gagcgttcctgggtgcctgt gtggctgggt 120 ctgatgagcc tggcccagag ggcctcacct ccacctccctgctagacctc ctgctgccca 180 ctggcttgga gccactggac tcagaggagc ctagtgagaccatgggcctg ggagctgggc 240 tgggagcccc tggctcaggc ttccccagcg aagagaatgaagagtctcgg attctgcagc 300 caccacagta cttctgggaa gaggaggaag agctgaatgactcaagtctg gacctgggac 360 ccactgcaga ttatgttttt cctgacttaa ctgagaaggcaggttccatt gaagacacta 420 gccaggctca agagctgcca aacctcccct ctcccttgcccaagatgaat ctggttgagc 480 ctccctggca tatgcctccc agagaggagg aagaagaggaagaggaagag gaggagaggg 540 agaaggaaga ggtagagaaa caagaggagg aggaagaggaggagctgctc cctgtgaatg 600 gatcccaaga agaagccaag cctcaggtcc gtgacttttctctcaccagc agcagccaga 660 ccccaggggc caccaaaagc aggcatgaag actccggggaccaggcctca tcaggtgtgg 720 aggtggagag cagcatgggg cccagcttgc tgctgccttcagtcacccca actacagtga 780 ctccggggga ccaggactcc accagccaag aggcagaggccacagtgctg ccagctgcag 840 ggcttggggt agagttcgag gctcctcagg aagcaagcgaggaagccact gcaggagcag 900 ctggtttgtc tggccagcac gaggaggtgc cggccttgccttcattccct caaaccacag 960 ctcccagtgg ggccgagcac ccagatgaag atccccttggctctagaacc tcagcctctt 1020 ccccactggc ccctggagac atggaactga caccttcctctgctaccttg ggacaagaag 1080 atctcaacca gcagctccta gaagggcagg cagctgaagctcaatccagg ataccctggg 1140 attctacgca ggtgatctgc aaggactgga gcaatctggctgggaaaaac tacatcattc 1200 tgaacatgac agagaacata gactgtgagg tgttccggcagcaccggggg ccacagctcc 1260 tggccctggt ggaagaggtg ctgccccgcc atggcagtggccaccatggg gcctggcaca 1320 tctctctgag caagcccagc gagaaggagc agcaccttctcatgacactg gtgggcgagc 1380 agggggtggt gcccactcaa gatgtccttt ccatgctgggtgacatccgc aggagcctgg 1440 aggagattgg catccagaac tattccacaa ccagcagctgccaggcgcgg gccagccagg 1500 tgcgcagcga ctacggcacg ctcttcgtgg tgctggtggtcattggggcc atctgcatca 1560 tcatcattgc gcttggcctg ctctacaact gctggcagcgccggctgccc aagctcaagc 1620 acgtgtcgca cggcgaggag ctgcgcttcg tggagaacggctgccacgac aaccccacgc 1680 tggacgtggc cagcgacagc cagtcggaga tgcaggagaagcaccccagc ctgaacggcg 1740 gcggggccct caacggcccg gggagctggg gggcgctcatggggggcaag cgggaccccg 1800 aggactcgga cgtgttcgag gaggacacgc acctgtgagcgcagcgaggc gcaggccgag 1860 tgggccgcca ggaccaagcg aggtggaccc cgaaacggacggcccggagc ccgcaccagc 1920 cccgcgccta cccggccgcc cccgcgcctg gccctcggcgcgggctcctt cccgcttccc 1980 ccgacttcac acggcggctt cggaccaact ccctcactcccgcccgaggg gcaggcctca 2040 aagcccgcct tggccccgct ttcccgcccc tgaaccccggccccgcgggc ggcgggcgcg 2100 cttcctgcgc cccgggactc aattaaaccc gcccggagaccacgcgggcc cagcgaaaaa 2160 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2220 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaa 2269 2 605 PRT human 2 Met Gly Arg Leu Leu Arg Ala Ala Arg LeuPro Pro Leu Leu Ser Pro 1 5 10 15 Leu Leu Leu Leu Leu Val Gly Gly AlaPhe Leu Gly Ala Cys Val Ala 20 25 30 Gly Ser Asp Glu Pro Gly Pro Glu GlyLeu Thr Ser Thr Ser Leu Leu 35 40 45 Asp Leu Leu Leu Pro Thr Gly Leu GluPro Leu Asp Ser Glu Glu Pro 50 55 60 Ser Glu Thr Met Gly Leu Gly Ala GlyLeu Gly Ala Pro Gly Ser Gly 65 70 75 80 Phe Pro Ser Glu Glu Asn Glu GluSer Arg Ile Leu Gln Pro Pro Gln 85 90 95 Tyr Phe Trp Glu Glu Glu Glu GluLeu Asn Asp Ser Ser Leu Asp Leu 100 105 110 Gly Pro Thr Ala Asp Tyr ValPhe Pro Asp Leu Thr Glu Lys Ala Gly 115 120 125 Ser Ile Glu Asp Thr SerGln Ala Gln Glu Leu Pro Asn Leu Pro Ser 130 135 140 Pro Leu Pro Lys MetAsn Leu Val Glu Pro Pro Trp His Met Pro Pro 145 150 155 160 Arg Glu GluGlu Glu Glu Glu Glu Glu Glu Glu Glu Arg Glu Lys Glu 165 170 175 Glu ValGlu Lys Gln Glu Glu Glu Glu Glu Glu Glu Leu Leu Pro Val 180 185 190 AsnGly Ser Gln Glu Glu Ala Lys Pro Gln Val Arg Asp Phe Ser Leu 195 200 205Thr Ser Ser Ser Gln Thr Pro Gly Ala Thr Lys Ser Arg His Glu Asp 210 215220 Ser Gly Asp Gln Ala Ser Ser Gly Val Glu Val Glu Ser Ser Met Gly 225230 235 240 Pro Ser Leu Leu Leu Pro Ser Val Thr Pro Thr Thr Val Thr ProGly 245 250 255 Asp Gln Asp Ser Thr Ser Gln Glu Ala Glu Ala Thr Val LeuPro Ala 260 265 270 Ala Gly Leu Gly Val Glu Phe Glu Ala Pro Gln Glu AlaSer Glu Glu 275 280 285 Ala Thr Ala Gly Ala Ala Gly Leu Ser Gly Gln HisGlu Glu Val Pro 290 295 300 Ala Leu Pro Ser Phe Pro Gln Thr Thr Ala ProSer Gly Ala Glu His 305 310 315 320 Pro Asp Glu Asp Pro Leu Gly Ser ArgThr Ser Ala Ser Ser Pro Leu 325 330 335 Ala Pro Gly Asp Met Glu Leu ThrPro Ser Ser Ala Thr Leu Gly Gln 340 345 350 Glu Asp Leu Asn Gln Gln LeuLeu Glu Gly Gln Ala Ala Glu Ala Gln 355 360 365 Ser Arg Ile Pro Trp AspSer Thr Gln Val Ile Cys Lys Asp Trp Ser 370 375 380 Asn Leu Ala Gly LysAsn Tyr Ile Ile Leu Asn Met Thr Glu Asn Ile 385 390 395 400 Asp Cys GluVal Phe Arg Gln His Arg Gly Pro Gln Leu Leu Ala Leu 405 410 415 Val GluGlu Val Leu Pro Arg His Gly Ser Gly His His Gly Ala Trp 420 425 430 HisIle Ser Leu Ser Lys Pro Ser Glu Lys Glu Gln His Leu Leu Met 435 440 445Thr Leu Val Gly Glu Gln Gly Val Val Pro Thr Gln Asp Val Leu Ser 450 455460 Met Leu Gly Asp Ile Arg Arg Ser Leu Glu Glu Ile Gly Ile Gln Asn 465470 475 480 Tyr Ser Thr Thr Ser Ser Cys Gln Ala Arg Ala Ser Gln Val ArgSer 485 490 495 Asp Tyr Gly Thr Leu Phe Val Val Leu Val Val Ile Gly AlaIle Cys 500 505 510 Ile Ile Ile Ile Ala Leu Gly Leu Leu Tyr Asn Cys TrpGln Arg Arg 515 520 525 Leu Pro Lys Leu Lys His Val Ser His Gly Glu GluLeu Arg Phe Val 530 535 540 Glu Asn Gly Cys His Asp Asn Pro Thr Leu AspVal Ala Ser Asp Ser 545 550 555 560 Gln Ser Glu Met Gln Glu Lys His ProSer Leu Asn Gly Gly Gly Ala 565 570 575 Leu Asn Gly Pro Gly Ser Trp GlyAla Leu Met Gly Gly Lys Arg Asp 580 585 590 Pro Glu Asp Ser Asp Val PheGlu Glu Asp Thr His Leu 595 600 605

What is claimed is:
 1. Podocalyxin-like protein having selectin bindingactivity.
 2. The podocalyxin-like protein according to claim 1, whereinsaid protein is a glycoprotein.
 3. The podocalyxin-like proteinaccording to claim 2, wherein said protein is derived from highendothelial venules.
 4. The podocalyxin-like protein according to claim3, wherein said protein is derived from tonsilar high endothelialvenules.
 5. A selectin ligand having the binding activity of thepodocalyxin-like protein derived from high endothelial venules.
 6. Theligand according to claim 5, wherein said ligand is a ligand forL-selectin.
 7. The ligand according to claim 5, wherein said ligand is aligand for P-selectin.
 8. The ligand according to claim 5, wherein saidligand is a glycoprotein.
 9. The ligand according to claim 5, whereinsaid ligand comprises an amino acid sequence found in humanpodocalyxin-like protein.
 10. The ligand according to claim 5, whereinsaid amino acid sequence is found in the membrane distal region of theprotein.
 11. A pharmaceutical composition comprising an active agentthat modulates the selectin binding activity of podocalyxin-likeprotein.
 12. The pharmaceutical composition according to claim 11,wherein said active agent is an agent that inhibits an enzymeresponsible for post-translational modification of said protein, wheresaid post-translational modification results in the selectin bindingactivity of said protein.
 13. The pharmaceutical composition accordingto Claim 11, wherein said active agent modulates the expression of saidpodocalyxin-like protein.
 14. The pharmaceutical composition accordingto claim 11, wherein said active agent is a selectin ligand having thebinding activity of the podocalyxin-like protein derived from highendothelial venules.
 15. The pharmaceutical composition according toclaim 14, wherein said selectin ligand is a P-selectin ligand.
 16. Thepharmaceutical composition according to claim 14, wherein said selectinligand is an L-selectin ligand.
 17. A method for inhibiting a bindingevent between a selectin and podocalyxin-like protein, said methodcomprising: contacting said selectin with a selectin ligand having thebinding activity of podocalyxin-like protein.
 18. The method accordingto claim 17, wherein said selectin is P-selectin.
 19. The methodaccording to claim 17, wherein said selectin is L-selectin.
 20. Themethod according to claim 17, wherein said binding event is an in vitrobinding event.
 21. The method according to claim 17, wherein saidbinding event is an in vivo binding event.
 22. A method of inhibiting aselectin mediated binding event in a mammalian host, said methodcomprising: administering to said host an effective amount of apharmaceutical composition comprising an active agent that modulates theselectin binding activity of podocalyxin-like protein.
 23. The methodaccording to claim 22, wherein said active agent inhibits an enzymeresponsible for post-translational modification of said protein, wheresaid post-translational modification results in the selectin bindingactivity of said protein.
 24. The method according to claim 22, whereinsaid active agent modulates the expression of said podocalyxin-likeprotein.
 25. The method according to claim 22, wherein said active agentis a selectin ligand having the binding activity of the podocalyxin-likeprotein derived from high endothelial venules.
 26. The method accordingto according to claim 25, wherein said selectin ligand is a P-selectinligand.
 27. The method according to claim 25, wherein said selectinligand is an L-selectin ligand.
 28. A method of modulating a symptom ina mammalian host of a disease condition associated with a selectinmediated binding, said method comprising: administering to said host apharmaceutical composition comprising an effective amount of an activeagent that modulates the selectin binding activity of high endothelialvenule podocalyxin-like protein.
 29. The method according to Claim 28,wherein said symptom is inflammation.
 30. The method according to claim28, wherein said disease condition is selected from the group consistingof acute inflammation, chronic inflammation, autoimmune disease, tissuerejection, atherosclerosis, restinosis, and damaging thombotic event.